Bioprocess Technology(Operation Modes and Scales)

13th. July 2010CEPP, UTM Skudai, Johor

Prof. Dr. Hesham A. El EnshasyFaculty of Chemical Engineering

CEPP, UTM, Skudai, Malaysia

1- Two-Phases vs. Three-Phases system

2- Free vs. Immobilized cell system

3- Living cell and enzyme system

Cultivation systems in Bioprocess Industries

1- Closed system (Batch culture)

2- Semi-closed system (Fed-batch culture)

3- Opened system (Continuous culture).

Other modes:- Immobilized cells

A- Repeated batch cultureB- Continuous culture

- Perfusion culture

Cultivation modes of submerged culture

Operation principle (A) and kinetics (B) of cell growth, nutrient consumption, and production formation during batch,

fed-batch, and continuous operation.

Batch cultivation:

Batch culture is a closed culture system which contains an initial, limited amount of nutrient. The inoculated culture will pass through a number of phases as follows:

•Lag Phase •Log Phase (exponential growth phase)•Stationary Phase (stagnant phase, maximum population phase)•Decline Phase (death phase).

I II III IV

Typical Microbial Growth curve

I- The Lag PhaseThis phase can be described as an adaptation phase of the cell for the new environment. The length of lag phase depends on the changes in nutrients composition of the new medium and on the age of inoculum. In bioprocess design, it is necessary to minimize the length of lag phase in order to obtain maximum utilization of the bioreactor. Therefore, the following points should be considered:

1- The inoculum should be active as possible (preferably in the exponential growth phase).

2- The medium used to grow the inoculum should correspond as closely as possible to the medium to be used in the large scale bioreactor.

3- A reasonably large volume of inoculum should be used (not less than 5% of the working volume of the bioreactor).

Typical Microbial Growth curve

II- The Log Phase:

During this phase, cells grow exponentially with time. The relation between time and cell growth during this phase can be described simply as follows:

Xdtdx

Where, X, is the concentration of microbial biomass, t, is time in hours and µ, is the specific growth rate in [h-1].

In general, it is easy to visualize the exponential growth of unicellular organisms which replicate by binary fission. Also, animal and plant cells in suspension culture behave very similar to unicellular microorganisms.

Typical Microbial Growth curve

µmax (the maximal specific growth rate) of different group of organisms

Organism µmax [h-1]

Vibrio natriegens 4.24

Methylomonas methanolytica 0.53

Penicillium chrysogenum 0.12

Fusarium graminearum 0.28

Plant cells (in cell culture) 0.01-0.046

Animal cells (in cell culture) 0.01-0.05

III-Stationary Phase

During this phase, the change in cell mass with time kept constant. This may due to either the rate of growth is equal to cell death or the termination of cell reproduction with no cell death.

Why cells enter stationary phase ?

How long is this phase ?

Do cell needs energy during this phase ?

Typical Microbial Growth curve

IV- Decline Phase (death phase)

This phase is characterized by significant decrease in cell mass (cell number) due to cell lysis.

Typical Microbial Growth curve

In Bioprocess point of view, the change in biomass value can be described simply during different phases of batch culture as follows:

vedtdx

dtdx

dtdx

vedtdx

phasedecline

phasestationaryphaselag

phase

.

..

.log

0

Basic types of product formation kinetics during batch operation. spec: Specific growth rate; qspec: specific production rate.

Growth of filamentous microorganisms

In submerged cultivation involving filamentous organisms, the

morphology can vary from discrete compact pellets of hyphae to

homogeneous suspension of dispersed mycelia. These

morphological differences are associated with significant differences

in growth kinetics and physiology. Growth of dispersed mycelia is

effectively equivalent to that of unicellular, with homogenous

distribution of biomass, substrates and products and exponential

growth (Monod type) at a constant specific growth rate in batch

culture where substrate(s) are in excess.

In the case, the biomass is represented as a sphere

M = (Sphere volume Density)

M = 43 / 3

In case of growth in pellet form, the microbial growth is affected by pellet morphology. This gives two extremes. In case of pellet consists of densely packed hyphae, growth is restricted by diffusion of material from the liquid phase to the pellet centre and the growth is limited to the hayphae in the outer peripheral shell. Thus, in batch culture, the biomass (M) increases as cubic function of time.

3/10

3/1 MktM

Where M0 represents the initial biomass and k is a constant.

Growth of filamentous microorganisms

Layer A

Layer B

Layer C

Layer D

Hollow core of pellet

(A) The external layers of fungal pellet (bar = 300

µm). (B) Pellet core showing layers C, D and the

hollow core of pellet

(bar = 300 µm). (C ) A closer look to the central part

of pellet (bar = 40 µm).

Schematic diagram of fungal pellet insubmerged cultures

If a culture assumed to constant of n spherical pellets, of equal radius r and density , with an active outer mycelial shell of width w, growing at a specific rate µ, then the constant k (the rate by which pellet radius increase due to growth) can be determined as follows:

wnk ...34 3/1

Growth of filamentous microorganisms

Parameter Filamentous growth Pelleted growth

Growth rate Hi Low

Rheological behaviour Non-Newtonian

(Viscous)

Newotonian

(non viscous)

Mass transfer

(oxygen/substrate/product)

1. In the bioreactor

2. In the microbial system

Low

Hi

Hi

Low

Operational condition Cause many

problems

Easy to be controlled

Pellet form vs. Filamentous form

0 20 40 60 80 100 120

0

2

4

6

8

10

12

CD

W [g

l-1]

time [h]

0

50

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250

300(B)

GO

x(to

tal)

[µka

t l-1]

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gluc

ose

[g l-1

]

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]

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l-1]

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x(to

tal)

[µka

t l-1]

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gluc

ose

[g l-1

]

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PO

2 [%

]

Filamentous Growth

Pellet Growth

Batch cultivation of Aspergillus niger in small scale bioreactor using glucose as sole C-source. (A), Growth in small aggregate-filamentous form. (B), growth in pellet form.

0 5 10 15 20 25 30 35 40

0

50

100

150 (C) filamentouspellet

time [h]

QO

2 [mm

ol l-

1 h-

1 ]

0

10

20

30 (B) filamentouspellet

QC

O2 [m

mol

l-1

h-1 ]

0

1

2

3(A) filamentous

pellet

RQ

0 10 20 30 40 50 80 900

20

40

60

80

100 filamentous

time [h]

(A)

Car

bon

cont

ent [

%]

0 10 20 30 40 50 80 900

20

40

60

80

100 Pellet(B)

Car

bon

cont

ent [

%]

time [h]

CDW, glucose, gluconate, oxalate, CO2

The differences in respiration activities and C-balance when cell grow in Filamentous- and pellet form

200 RPM

500 RPM

800 RPM

19 h 25 h

Macro-morphological growth of A. niger under different agitation speeds

33

22

34

234 wDDV totaltotal

active

w

D

Pellet-Morphology in 5-L bioreactor (200 rpm) after staining with AO

Fed-Batch Cultivation

Fed-batch cultivation is superior to conventional batch especially when changing concentrations of nutrient(s) affect the yield or productivity of the desired metabolite(s). There are also other minor advantages of medium feeding. However, these advantages can be summarized as follows:

1- Substrate inhibition

2- Catabolite repression

3- Extension of operation time

4- Replacement of water lost by evaporation

5- Decreasing viscosity of broth

6- High cell density cultivation

1- Substrate inhibition

Nutrients such as ethanol and aromatic compounds inhibit the

growth of microorganisms if added at the zero time of cultivation.

By addition of these substrate(s) by fed-batch cultivation strategy,

lag-time can be shortened and the inhibition of cell growth

significantly reduced.

Why Fed-Batch Cultivation ?

2- Catabolite repression

When a microorganism is provided with a rapidly metabolized carbon-energy source such as glucose, the resulting increase of the intracellular concentration of ATP leads to the repression of enzyme synthesis, thus causing a slower metabolization of the energy source. This phenomena is known as catabolite repression. A powerful method to overcoming catabolite repression in enzyme biosynthesis is a fed-batch culture in which the glucose concentration in the culture liquid is kept low, where growth is restricted, and enzyme synthesis is depressed.

Why Fed-Batch Cultivation ?

3- Extension of operation time

In a non-growth-associated microbial process, such as antibiotic production, microorganisms initially rapidly utilize the carbon-energy source for growth and then synthesize the desired secondary metabolite in the subsequent declining phase and early stationary phase.

In the conventional batch process, this production phase is short, due to the depletion of the carbon-energy source; the subsequent cell autolysis is rapid and severe. Thus, after transition from growth to production phase, it is important to maintain a concentration of the carbon-energy source where the microorganisms are semi-starved but where enzyme activity for synthesis is highest.

Why Fed-Batch Cultivation ?

4- Replacement of water lost by evaporation

In aerobic microbial processes during extended reaction period, such as in antibiotic production (1-2 weeks), considerable amounts of water are lost as the vapour from through exhaust gas. For example for a cultivation process operation at 30°C with 1.0 vvm aeration (60% relative humidity), about 25% of water will be lost after 2 weeks. This leads to a considerable concentration of the mycelial broth and an accompanying changed in its rheological behaviour.

Why Fed-Batch Cultivation ?

5- Decreasing viscosity of broth

In microbial biopolymer production such as dextran, pullulan and xanthan, broth viscosity can be kept low by continuous feeding of nutrients. Otherwise, the significant increase in broth viscosity will raise the agitation power consumption and low oxygen transfer efficiency.

Why Fed-Batch Cultivation ?

6- High cell density cultivation

To achieve high cell density concentration (some times up to 100 g CDW per liter) in batch culture, a high concentration of nutrients is required. As such high concentrations nutrients become inhibitory. Thus, fed-batch cultivation is necessary to achieve a high cell density culture.

Why Fed-Batch Cultivation ?

Types of Fed-batch Cultivation Strategies

Without Feeback control With Feeback control

1- Intermittent addition2- Constant rate3- Exponential increase rate4- Optimized5- Others

1- Indirect feedback control2- Direct feedback control3- Constant-value control4- Optimal control

Type of Feeding and metabolite production

0 50 100 150 200 250 300 350 4000123456789

0.0

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CD

W [g

L-1]

time [h]

E

PS

[ g

L-1]

intermittent glucose addition [90 g ]

g

luco

se [g

L-1]

D

O [%

]

O

ut-g

as O

2 [%

]

o

ut-g

as C

O2 [

%]

0 50 100 150 200 250 300 350 4000123456789

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.50

5

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CD

W [g

L-1]

time [h]

E

PS

[ g

L-1]

g

luco

se [g

L-1]

D

O [%

]

O

ut-g

as O

2 [%

]

Fed-Batch PhaseBatch Phase

o

ut-g

as C

O2 [

%]

Cell growth and EPS production in fed-batch culture. Arrow show the time at which glucose was fed to the bioreactor in single shot addition

Cell growth and EPS production by in fed-batch culture in CO2 stat culture. Glucose was fed to keep constant concentration of carbon dioxide in out-gas of the bioreactor

Example:Fed-batch cultivation strategy (exponential feeding) for a recombinant strain of Asperigllus niger for glucose oxidase production.

)(.).(..1)(/

FttseteXtVmY

tm FLEsetSX

s

WhereMs Mass flow of substrate [g h-1]t Cultivation time [h]tF Start time of feeding phase [h]µset Adjusted specific growth rate [h-1]E Maintenance coefficient [g g-1 h-1]YX/S The biomass/substrate yield coefficient [g g-1]XF The biomass concentration at the start time of feeding phase [g]VL The culture volume [L]

Exponential feeding of substrate(s)

3- Open system (Continuous Culture)

In continuous cultivation strategy, the substrate is added to the bioreactor continously at a fixed rate. This maintains the organisms in the logarithmic growth phase. The fermentation products are taken out continuously. The design and arrangements for continuous fermentation, are some what complex.

Common strategies for continuous culture

A- Chemostat Culture : Key nutrient concentration kept constant during the process (growth rate is controlled by dilution rate (D)

B- Turbidostate: (Optical density of culture kept constant during the process)

In chemostat culture, nutrients are supplied at a constant flow rate and the cell density is adjusted with the supplied essential nutrients for growth. Thus, growth rate is determined by the utilization of substrates such as: Carbon, nitrogen and phosphate.

Simple Continuous culture (Chemostat Mode)

Biomass Balance in Continuous culture

Continuous culture: Growth at steady state condition

Advantages of Continuous Culture

Immobilized cell system

1- Increase cell density to high level

2- Higher yield based on inceasing enzyme stability

3- Operation under continuous and repeated batch mode with high yield

4- Reduce the production time (especially for secondary metabolites)

5- Protect cells from shear stress effect (example: Mammalian and plant cells).

6- Reduce the cost of medium

7- Long term operation with low preparation time

8- Ease down stream process (Cell separation steps)

9- Increase genetic stability in case of using recombinant strain

Advantages

Immobilized cell system

1- Cost

2- By products Removal

3- Oxygen/Carbon dioxide diffusion

4- Substrate(s) diffusion

5- Growth rate determination

Disadvantages

Immobilized cell system

Adsorption Entrapment

Saw dust

Glass wool

Alginate

Carrageenan

Glass wool treated with PEI prior cell immobilization

Main Methods of Cell Immobilization

Kinetics of cell growth and gluconic acid production of a recombinant strain of A. niger (GOD 3-18). Closed and opened symbols represent the free and immobilized cultures, respectively.

0 10 20 30 40 50 60 70 80 90 10002468

1012141618

020406080

100120140160180

020406080100120140160

-202468

101214161820

0,00,20,40,60,81,01,21,41,61,82,0

CD

W [g

/l]

Time [h]

g

luco

se [g

/l]

g

luco

nic

acid

[g/l]

Y

[P/X

] [g

gluc

onat

e/ g

cel

ls]

Y

[P/S

] [g

gluc

onat

e/ g

glu

cose

]

Immobilized cells on GW treated with PEI showed no effect on the production of GA

Immobilized cells have higher specific production

The fermentation medium used for gluconic acid production By immobilized cells was of the following composition [g/l]:

Complete medium Minimal medium

glucose, 160.0 160.0NaNO3, 3.0 1.0K2HPO4, 1.0 -MgSO4.7H2O, 0.5 0.2KCl, 0.5 -FeSO4.7H2O, 0.01 -Yeast extract, 2.0 -

The pH of medium was adjusted to 5.5

0 1 2 3 4 5 6 7 8 9 100

20

40

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140

160

180

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220

240

gluc

onic

aci

d [g

/l]

Batch No.

complete medium

*

minimal medium

Repeated batch cultivation of immobilized spores of a recombinant A. niger In both complete and minimal medium in batch time of 24 h.

(*), the first batch was cultivation in complete medium for 48 h in both cases.

Comparison between cultivation parameters for wild type and r A. niger in both batch and repeated batch cultures.

Microorganism and Cultivation method

Initial glucose

conc. [g/l]

Xmax

[g/l]Pmax

[g/l]Qp

[g/lh]Y [P/X]

[g/g]tc

[h]

Recombinant A. nigerBatch, Free cells

160 12,55 136 2,83 10,84 48

Recombinant A. nigerBatch, Immobilized cells

160 8,90 140 2,92 15,73 48

Recombinant A. nigerRepeated batchcomplete medium

160 - 156 6,50 - 24

Recombinant A. niger Repeated batchminimal medium

160 - 154 6,42 - 24

Abbreviations:Xmax: maximal cell dry weight; Pmax: maximal gluconic acid production, Qp: volumetric gluconic acid production rate, tc: Cultivation time.

0 20 40 60 80 100 1200

2

4

6

8

10

120

1

2

3

4

0

50

100

150

200

250

Cel

l num

ber (

*105 )

time [h]

Living Dead Total

Glucose Lactate

Glu

cose

/Lac

tate

[g L

-1]

MA

b [ m

g L-1

]

0 50 100 150 200 250 3000.00.20.40.60.81.01.21.41.61.82.00

1

2

3

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5

6

0

50

100

150

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350VIIIVIIVIVIVIIIIII

Cel

l num

ber (

*105 )

time [h]

Living Dead Total

Glucose Lactate

Glu

cose

/Lac

tate

[g L

-1]

MA

b [ m

g L-1

]

Efficient Monoclonal Antibody Production in basket Spinner Free vs. Immobilized Cells

MAb production using free cells (batch mode) MAb production using immobilized cells (repeated batch mode)

oxygen oxygen nutrients

Spent medium

Schematic batch culture and perfusion cultures: Mammalian cells

Cell Density Low High

System productivity Low High

Lactate inhibition effect High Low

cell

inhibitor

product

Thank You